Stress induced TDP-43 mobility loss independent of stress granules

TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting “crucibles” for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration. ‘Stimulated-emission depletion microscopy’ (STED) and ‘tracking and localization microscopy’ (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. This work provides insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization and aggregation takes place in the cytoplasm separate from SGs.


REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this interesting paper from Streit et al., the authors utilize advanced single particle tracking and superresolution microscopy to characterize the mobility and the aggregation of TDP-43, an important protein that has been shown to localize in aggregates in neurodegenerative diseases such as amyothropic lateral sclerosis. Upon stress, TDP-43 is known to accumulate in stress granules (SG), but it is currently unknown, whether recruitment to SG is a protective or a promoting event towards pathological aggregation is currently debated.
The authors show that, upon stress TDP-43 molecules slow down, and such slow down is not limited to stress granules, but rather widespread in the nucleus and in the cytoplasm. Recovery from stress results in a recovery of TDP-43 mobility only when the stress is mantained for < 1hr.
Further the authors show that TDP-43 localization in stress granules is not homogeneous, that different TDP-43 molecules can revisit the same site on SG multiple times, and that TDP-43 can also localize ad diffuse on the surface of unidentified vescicular structures.
Overall the paper is interesting and well written, and the methods have been carried out with rigor and with the necessary controls. As detailed below, I believe that it is maybe not surprising to observe a global slow-down of TDP in conditions leading to TDP-43 aggregation. Nevetheless, I think that the presented results are interesting because they hint to the possibility that aggregation of TDP-43 can occur at sites other than SGs, with potential implications for the mechanism underlying the spreading of ALS phatogenesis.
I also liked that the authors have limited their discussion to describe their data in the context of the avaiable literature, without excessive speculations. Nevetheless, I think that some additional experiments could increase the impact of the presented results, as I will detail below, together with some minor comments on some technical details.
1. While I agree that the observation that TDP-43 displays globally reduced mobility upon stress is one of the most important results of the paper, this does not appear to me as surprising as the authors present it. As the authors mention in the discussion, sodium arsenite (the stress source used throughout the manuscript) causes ATP depletion, and it is expected that ATP depletion result in a decrease of mobility of many cellular factors undergoing transient interactions (e.g. transcription factors, PMID: 15189848, PMID: 15024032, RNA binding proteins PMID: 12473688, epigenetic factors: PMID: 34313222 etc). This raises the question on wheter other stress sources (maybe not associated to reduction of intracellular ATP levels) can induce a similar slow down on TDP-43, and whether other aggregating proteins involved in ALS (FUS?) show a similarly global slow-down upon stress induction. I understand that these experiments might fall beyond the scope of this paper, but they would strongly direct the interpretation of the presented data, in my opinion.
2. It is interesting that TDP mobility in stress granules is comparable to the slowest average mobility observed in the cytoplasm at late time points. ( Figure 3C). Is at this time point a dynamic shuttling of TDP-43 in and out of SGs? Have the authors tried to analyze how frequently single TDP-43 molecules get trapped/escape stress granules at different timepoints. This might point to TDP acquiring more solid-like properties upon 'chronic' stress, also providing an explanation for their 'recovery' experiments.
3. Related to this, it was unclear to me whether the recovery experiment was performed on the whole cytoplasm (including SGs) or only in the cytosolic fraction. It would be interesting to see whether TDP-43 is more refractory to recoverying when incorporated in SGs. Fig 4B, it looks like the cytosolic patches in which TDP-43 show low mobility are larger than the hotspots observed in SG. The impression from these crops, is that while in SG TDP is highly mobile, with the exception of the binding hot-spots, in these cytosolic patches the TDP-43 diffusion is slowed down, and maybe rendered more anomalous, potentially trapping TDP-in these regions. It would be very interesting to monitor the anomaly of diffusion in these patches, to check whether these zones trap TDP. One simple approach could be to analyze the anisotropy of displacements as in Hansen et al., 2020 PMID: 31792445).

Talm analysis. For the insets in
5, The figure about the 'vescicle' is very interesting, but it raises two questions: a. How frequent are these observations. b. What is the substrate there? If these events are sufficiently frequent, maybe the authors could try to perturb possible candidates (lysosomes, large endosomes, others?), to try to figure it out.

Minor-points:
-In the discussion the authors speak about TDP-43 oligomerization as the cause for its slowdown. but i think that aggregation would be a better term. Indeed, the current data could be explained both by the formation of large (homo)oligomers, but also by interactions with scaffolds and non-soluble cytosolic components.
-The authors have chose to only account the first 5 displacements for their analysis of TDP diffusion. I am aware that this has been proposed before by Hansen, 2018 to avoid counting of the slowest molecules (that remain in focus for longer), but the choice of the max track length always seemed arbitrary to me, and it is easy to show that this can have the net effect of under-representing the immobile population. It's not a big deal because the authors mostly focus on relative differences, however it would be advisable to use other methods to correct for disappearance of molecules going out of focus (e.g. explicitly correcting the different populations for their probability of going out of focus).
-In Figure S.1 the IF controls Fields of view are the same as the ones used in Figure 1. It would be advisable to either choose different FOVs or specify in the figure legend that the same FOV were usedto avoid being potentially notified for 'duplications'.
-Numbering of the last two supplementary figures is off.
-The numbers on the x-axis of Fig 4 A, C , E are in the wrong order.
-Line 462 (third paragraph in the discussion). The sentence is incomplete. Maybe "during prolonged stress"? In SGs?
Reviewer #2 (Remarks to the Author): In this manuscript, Streit et al. analyse the mobility and localization of TDP-43 under different stress conditions. For most of the experiments, they relied on a cell line moderately overexpressing Halotagged TDP-43. A major finding of the study is that the mobility of cytoplasmic TDP-43-Halo changes at stress conditions, which suggests that TDP-43 oligomerization occurs already in the cytoplasm independent of stress granules. This is an interesting finding that merits publication.
However, I do have concerns on the way the data are presented.
Throughout the manuscript, I am missing information on the size of the data set that has been analysed. For example, I did not see any information on the number of cells and single molecule tracks that have been analysed.
In the Reporting Summary the authors state that "the exact sample size for each experimental group/condition is given as a discrete number…". I did not find this information in the manuscript.
The authors also state in the same Reporting Summary "The number of cells subjected to tracking analysis was calculated (data available upon request)". For me, this is a very unreasonable statement. Why is the number of cells analysed not given?
This information (the number of analysed cells; the number of tracks; experimental repeats) should be provided in the manuscript. Without such information, it is hardly possible to validate the reported experimental results.
Figures 5, 6, and 7 entirely lack a statistical analysis. This is not sufficient.
For example, Fig. 5 shows just one STED-image of a single cell for each condition. Presumably, these are representative images, but without an unbiased quantification, it is just not possible to draw any conclusion from these images.
There needs to be quantification of the observations reported in Figs. 5, 6 and 7.
Reviewer #3 (Remarks to the Author): In the manuscript "Stress induced TDP-43 mobility loss independent of stress granules", the authors generated transgenic lines expressing N-and C-terminally HALO-tagged TDP-43 to investigate the mobility of the tagged proteins under oxidative stress conditions in the cytoplasm, stress granules, and the nucleus, using live cell single-molecule tracking and STED super-resolution microscopy. The authors observed stress induced decrease of TDP-43 mobility in all compartments suggesting that TDP-43 oligomerization may take place in the cytoplasm separate from SGs.
A potential role of stress granules for the pathological phase transition of TDP-43 and other proteins containing intrinsically disordered regions undergoing LLPS is of interest and still not well understood.
However, the results are difficult to interpret and do not provide significant new insights into the pathophysiology of TDP-43 for the broad readership of this journal, as discussed in more detail below. 1) Adding more confusion than clarity, the authors interpret decreased mobility as changes in oligomerization in the context of pathology. (This is problematic by itself since other protein or RNA interactions may have a similar effect). However, homo-oligomerization of TDP-43 occurs under normal physiological conditions and is required for performing its RNA regulatory functions, such as mRNA splicing. This may be related but is different from pathological aggregation.
2) It is unclear whether the effect of sodium arsenite on protein mobility is specific for TDP-43, or to proteins undergoing LLPS, or a very general effect of oxidative stress. A shut-down of protein synthesis, ATP depletion, and many other fundamental metabolic changes in the cell (as mentioned in the Discussion section) may potentially cause widespread differences in protein mobility with very unclear significance for TDP-43 pathophysiology. For all we know this may affect GFP and other random proteins to the same extent.
3) The neuroglioma cell lines overexpress 33kDa HALO-tagged TDP-43 constructs that change the properties of the proteins. The C-terminal tag leads to a significant mislocalization of TDP-43 into the cytoplasm. This may be due to increased fragmentation and generation of truncated constructs missing the NLS and parts of the RRM domains. This physiological oligomerization of TDP-43 requires its Nterminal domain, and RNA binding requires the RRM1 & 2 domain. It is unclear from the data what specific or mixed protein species are being investigated in SGs and the cytoplasm. It is also unclear how the shift in mobility relates to pathology-relevant features such as RNA-binding, PTMs (phospho-TDP-43), fragmentation, and oligomerization/aggregation state of TDP-43.
4) The Ctrl lanes in the Suppl. Figure S4 western blot for HALO-TDP-43 show a prominent 35kDa fragment, while the soluble and insoluble fractions do not.

Reviewer #1:
The reviewer states that "In this interesting paper from Streit et al., the authors utilize advanced single particle tracking and super-resolution microscopy to characterize the mobility and the aggregation of TDP-43, an important protein that has been shown to localize in aggregates in neurodegenerative diseases such as amyotrophic lateral sclerosis." and "I think that the presented results are interesting because they hint to the possibility that aggregation of TDP-43 can occur at sites other than SGs, with potential implications for the mechanism underlying the spreading of ALS pathogenesis." We appreciate that the reviewer is further positive about our work by stating "Overall the paper is interesting and well written, and the methods have been carried out with rigor and with the necessary controls." Nevertheless, the reviewer thinks "that some additional experiments could increase the impact of the presented results, as I will detail below, together with some minor comments on some technical details." We are very pleased with the overall positive assessment of our paper by Reviewer #1. We are also grateful for the specific points raised and suggestions that helped us improve the quality of the manuscript. understand that these experiments might fall beyond the scope of this paper, but they would strongly direct the interpretation of the presented data, in my opinion.

Reply:
We thank the reviewer for his/her helpful suggestion to study whether other stress sources, not associated to reduction of intracellular ATP levels, might induce a similar slow-down of TDP-43 Halo . Since sorbitol treatment has been shown to induce stress granule formation we applied sorbitol stress to our TDP-43 Halo cell culture model.
As also observed using sodium arsenite stress, sorbitol treatment significantly reduces TDP-43 Halo mobility in the cytoplasm as well as in the nucleus. This effect could also be detected analyzing the whole cell. The new data are presented as a new supplemental figure S8. Sorbitol stress leads to an overall strong reduction of TDP-43 Halo mobility, already in the first 20 min of stress. A similar effect was obtained for the HaloTag alone, pointing to a general shrinkage of the cell caused by the osmotic stress that has been reported previously (Munder et al., 2016).
Additionally, since ATP depletion was referred to by the reviewer as a possible source for the observed mobility slowdown of TDP-43 in presence of sodium arsenite, we performed additional experiments to test how much the intracellular ATP levels are reduced given our experimental conditions. We observed an insignificant reduction of ATP levels, that is most likely not sufficient to explain the strong mobility reduction observed for TDP-43 Halo under sodium arsenite stress. Moreover, we also want to refer to our control experiments of HaloTag alone, that did not show a slowdown with sodium arsenite stress duration (compare supplementary figure S6c) nor an increase in the relative are covered by cytoplasmic patches with stress duration (supplementary figure S19), thus lending further support to the interpretation that the observed effect is specific to TDP-43. This finding is in accordance with previous studies demonstrating that other proteins do not show a reduction of mobility upon energy depletion e.g. Furthermore, we agree with the reviewer that it would be highly interesting to study whether other aggregating proteins e.g. FUS would also show a general slow-down in the cytoplasm upon stress. However, we respectfully suggest that this might be subject of a future study since it falls beyond the scope of this paper. Reply: Following the reviewer's suggestion we determined shuttling events during sodium arsenite stress duration. We found that in the course of stress the number of shuttling events significantly decrease overall, but also when these events were normalized to the size of stress granules. Of note, when we analyzed the ratio of shuttling events in/out of stress granules, we observed a constant ration of around ~ 0.5. A detailed description and discussion of the new data is incorporated in the revised manuscript and the new data are presented as figure 2 c-e.
Point 3: Related to this, it was unclear to me whether the recovery experiment was performed on the whole cytoplasm (including SGs) or only in the cytosolic fraction. It would be interesting to see whether TDP-43 is more refractory to recovering when incorporated in SGs.

Reply:
We apologize that we presented the recovery data for the whole cell including SGs (former Fig. 5) in a way that was obviously not clear to the reviewer. Following the reviewers' suggestion, we have now analyzed TDP-43 Halo recovery in a region-specific manner and determined recovery of TDP-43 Halo mobility in the nucleus, in the cytoplasm and stress granules separately. The region-wise recovery data are in accordance with the data obtained for the whole-cell, showing that after 2h of stress TDP-43Halo mobility could not be fully regained after 4h of recovery. The new regionwise recovery data are displayed in supplementary figure S15. Fig 4B, it looks like the cytosolic patches in which TDP-43 show low mobility are larger than the hotspots observed in SG. The impression from these crops, is that while in SG TDP is highly mobile, with the exception of the binding hot-spots, in these cytosolic patches the TDP-43 diffusion is slowed down, and maybe rendered more anomalous, potentially trapping TDP-in these regions. It would be very interesting to monitor the anomaly of diffusion in these patches, to check whether these zones trap TDP. One simple approach could be to analyze the anisotropy of displacements as in Hansen et al., 2020 PMID: 31792445).  figure 7). Additionally, we analyzed TDP-43 Halo mobility within these structures and observed a mobility of ~ 0.5 µm 2 /s inside of these vesicle-like structures.

Point 4: TALM analysis. For the insets in
Although the observation of these vesicle-like structures is highly interesting, their rarity makes it very hard to determine a substrate especially during life cell tracking experiments, when only the transgenic proteins can be visualized. Since co-staining with e.g. LysoTracker were not applicable due to incompatibilities in the available wavelengths, several cell lines co-expressing vesicular markers would be needed to determine the substrate in a live-cell imaging based approach. We kindly suggest that this might be subject of future studies investigating TDP43 mobility within vesicular like structures.

Minor-points:
Point 6: In the discussion the authors speak about TDP-43 oligomerization as the cause for its slowdown, but I think that aggregation would be a better term. Indeed, the current data could be explained both by the formation of large (homo)oligomers, but also by interactions with scaffolds and non-soluble cytosolic components.

Reply:
We fully agree with the reviewer that we cannot distinguish between homooligomerization and/or the interaction/aggregation of TDP-43 with other components and we have clarified this in the revised discussion section. Moreover, to get further insights on the size of TDP-43 species under stress conditions we performed additional experiments, namely size-exclusion chromatography combined with TDP43-dot blotting. As shown in the new supplementary figure S5 application of 120 min of sodium arsenite stress clearly results in a shift of TDP43 from smaller species (fraction 84 -110 ml, corresponding to ~monomers) to bigger TDP-43 species (44 -74 ml factions corresponding to ~>10-mers). These results nicely complement our data on reduced mobility under stress conditions, however, we still cannot fully exclude that the shift in TDP-43 species is due to interactions to other proteins. We acknowledged this aspect in the revised version of the manuscript.

Point 7:
The authors have chosen to only account the first 5 displacements for their analysis of TDP diffusion. I am aware that this has been proposed before by Hansen, 2018 to avoid counting of the slowest molecules (that remain in focus for longer), but the choice of the max track length always seemed arbitrary to me, and it is easy to show that this can have the net effect of under-representing the immobile population.
It's not a big deal because the authors mostly focus on relative differences, however it would be advisable to use other methods to correct for disappearance of molecules going out of focus (e.g. explicitly correcting the different populations for their probability of going out of focus).

Reply:
As suggested by the reviewer we analyzed TDP-43 mobility data of the whole cell either using the first 5 or all displacements. The comparison clearly shows that the consideration of all displacements leads to an overall reduced mobility due to the stronger bias towards bound or slowly moving TDP-43 Halo species. Nevertheless, the observed effect of an overall stress-induced reduction of TDP-43 did not change. This additional analysis is incorporated in the revised version of the manuscript as supplemental Figure S4. Reply: We appreciate this comment, followed the suggestion of the reviewer and stated in the figure legend of supplementary figure S1 that we used the same FOV.
Since the image was taken from naïve H4 cells neither expressing C-or N-terminally tagged TDP-43 it serves as a control for both C-and N-terminally tagged TDP-43. Reply: We kindly appreciate this comment and corrected it in the revised manuscript.
Point 11: Line 462 (third paragraph in the discussion). The sentence is incomplete.
Reply: We apologize for this oversight and corrected it accordingly "prolonged stress in SGs".

Reviewer #2:
Also, reviewer#2 is positive about our work "This is an interesting finding that merits publication." However, the reviewer also has "concerns on the way the data are presented". Point 1: Throughout the manuscript, I am missing information on the size of the data set that has been analyzed. For example, I did not see any information on the number of cells and single molecule tracks that have been analyzed.

Reply:
We sincerely apologize for not providing this information. Lists with all analyzed cells per experimental condition and all found tracks are now given in the supplementary tables S1 and S2.

Point 2:
In the Reporting Summary the authors state that "the exact sample size for each experimental group/condition is given as a discrete number…". I did not find this information in the manuscript. The authors also state in the same Reporting Summary "The number of cells subjected to tracking analysis was calculated (data available upon request)". For me, this is a very unreasonable statement. Why is the number of cells analyzed not given? This information (the number of analyzed cells; the number of tracks; experimental repeats) should be provided in the manuscript. Without such information, it is hardly possible to validate the reported experimental results.

Reply:
We deeply apologize for this oversight. A list with all analyzed cells per condition is given in supplementary table S1. We also included the number of analyzed tracks in the supplementary table S2. Figures 5, 6, and 7 entirely lack a statistical analysis. This is not sufficient.

Point 2:
For example, Fig. 5 shows just one STED-image of a single cell for each condition.
Presumably, these are representative images, but without an unbiased quantification, it is just not possible to draw any conclusion from these images. There needs to be quantification of the observations reported in Figs. 5, 6 and 7. As already mentioned in our reply to Reviewer #1, we further analyzed TDP-43 anisotropy within these patches (as performed in Hansen et al., 2020 PMID: 31792445) and observed a strong anisotropy of TDP-43 Halo within these patches, indicating trapping and anomalous diffusion of TDP-43 Halo in these regions. We further looked at the diffusion coefficient of TDP-43 Halo within these cytoplasmic patches and observed a diffusivity of ~ 0.2 µm 2 /s (Figure 7 b, c).
Additionally, we analyzed and quantified TDP-43 binding hotspots or cluster as observed in stress granules and the cytoplasm. We observed an increasing cluster density within stress granules with increasing sodium arsenite stress, indicating a stress related clustering of TDP-43 Halo within stress granules (Figure 7d). In the cytoplasm, we observed a constant and low cluster density, indicating that cytoplasmic cluster of TDP-43 Halo are forming in a non-stress-related manner (Figure 7d

Reviewer #3:
The reviewer states "A potential role of stress granules for the pathological phase transition of TDP-43 and other proteins containing intrinsically disordered regions undergoing LLPS is of interest and still not well understood. "But the reviewer also states "the results are difficult to interpret and do not provide significant new insights into the pathophysiology of TDP-43 for the broad readership of this journal, as discussed in more detail below. " We are very grateful, that the reviewer agrees with us regarding the scientific interest into the general topic of the manuscript, and we are confident, that, given the additional experiments and data presented in the revised manuscript the importance of the novel data obtained becomes more evident.
Point 1: Adding more confusion than clarity, the authors interpret decreased mobility as changes in oligomerization in the context of pathology. (This is problematic by itself since other protein or RNA interactions may have a similar effect). However, homooligomerization of TDP-43 occurs under normal physiological conditions and is required for performing its RNA regulatory functions, such as mRNA splicing. This may be related but is different from pathological aggregation.

Reply:
The reviewer is of course right that homo-oligomerization of TDP-43 occurs under normal physiological conditions and is not necessarily related to pathological aggregation of TDP-43. As also stated in the response of point 6 of reviewer #1 we fully agree with the reviewer #3 that we cannot distinguish between homooligomerization and/or the interaction/aggregation of TDP-43 with other components and we have clarified this point in the revised manuscript. However, to get further insights on the size of TDP-43 species under stress conditions we performed sizeexclusion chromatography combined with TDP-43-dot blotting. As demonstrated as new supplemental figure S5 application of 120 min of sodium arsenite stress clearly results in a shift of TDP-43 from species eluting at fraction 84-110 ml corresponding to ~monomers to TDP-43 species eluting at 44-74 ml factions corresponding to >~10mers. These results nicely complement our data on reduced mobility under stress conditions, however, we still cannot fully exclude that that the shift in TDP-43 species is due to interactions to other proteins or cellular components. We acknowledged this aspect in the revised version of the manuscript. Furthermore, with the TDP-43 Halo cell line, we were able to track full-length, as well as fragmented TDP-43. The fragmented TDP-43 lacks the N-terminal domain required for dimerization. We therefore speculate that the observed mobility reduction might not solely caused by a functional dimerization of TDP-43.
Point 2: It is unclear whether the effect of sodium arsenite on protein mobility is specific for TDP-43, or to proteins undergoing LLPS, or a very general effect of oxidative stress.
A shut-down of protein synthesis, ATP depletion, and many other fundamental metabolic changes in the cell (as mentioned in the Discussion section) may potentially cause widespread differences in protein mobility with very unclear significance for TDP-43 pathophysiology. For all we know this may affect GFP and other random proteins to the same extent.

Reply:
We thank the reviewer for his/her helpful suggestion to study whether other stress sources not associated to reduction of intracellular ATP levels might induce a similar slow-down of TDP-43 Halo . Since sorbitol treatment has been shown to induce stress granule formation we applied sorbitol stress to our TDP-43 Halo cell culture model. In addition, given the reviewer's suggestion, we assessed the mobility of the HaloTag alone during sodium arsenite stress and did not observe a further reduction of mobility between 0-20 min and 100-120 min of sodium arsenite stress (supplementary figure   S6c). Furthermore, we analyzed and quantified cytoplasmic patches observed for TDP-43 Halo and HaloTag alone (Figure 7 and supplementary figure S19). For TDP-43 Halo we were able to observe the formation of cytoplasmic TDP-43 patches, exhibiting confined TDP-43 with a strongly reduced mobility and an increasing size of these TDP-43 patches with sodium arsenite stress duration. A similar effect could not be observed for the HaloTag alone (supplementary figure S19), indicating the observed effect is not a general stress-related effect.
In the revised manuscript we present additional data where we further assessed the aspect of ATP depletion during sodium arsenite stress, to relate potentially reduced ATP levels with the observed decrease in TDP-43 mobility. We observed an insignificant reduction of ATP levels (supplementary figure S9), that are most likely not sufficient to explain the strong mobility reduction observed for TDP-43 Halo under sodium arsenite stress. It was also shown for other proteins that their mobility is not reduced upon energy depletion e.g. (Munder et al., 2016), (Phair & Misteli, 2000), (Wagner, Chiosea, Ivshina, & Nickerson, 2004).

Point 3:
The neuroglioma cell lines overexpress 33kDa HALO-tagged TDP-43 constructs that change the properties of the proteins. The C-terminal tag leads to a significant mis-localization of TDP-43 into the cytoplasm. This may be due to increased fragmentation and generation of truncated constructs missing the NLS and parts of the RRM domains. This physiological oligomerization of TDP-43 requires its N-terminal domain, and RNA binding requires the RRM1 & 2 domain. It is unclear from the data what specific or mixed protein species are being investigated in SGs and the cytoplasm. It is also unclear how the shift in mobility relates to pathology-relevant features such as RNA-binding, PTMs (phospho-TDP-43), fragmentation, and oligomerization/aggregation state of TDP-43.

Reply:
We agree with the reviewer that for the C-terminally tagged cell line, we are not able to distinguish between full-length and fragmented TDP-43 in the course of the tracking analysis. However, we want to point out, that the N-terminally tagged TDP-43 cell line allows for the tracking of the full-length protein only. Supplementary figures S1 illustrates the establishment of the N-terminally tagged TDP-43 cell line. Tracking analysis using the N-terminally tagged cell line shows a similar stress-related and region specific reduction of TDP-43 mobility (Supplementary Figure S7). The TDP-43 Halo construct showed a slightly increased cytoplasmic mobility as compared to the Halo TDP-43 construct, indicating that a higher cytoplasmic mobility is most likely caused by fragmented TDP-43. We highlight in the revised version of the manuscript that the C-terminally tagged cell detects both truncated as well as full length TDP43.

Point 4:
The Ctrl lanes in the Suppl. Figure S4 western blot for HALO-TDP-43 show a prominent 35kDa fragment, while the soluble and insoluble fractions do not.

Reply:
The control lane is comprised of TDP-43 Halo cell lysates prepared by a different protocol and was used solely as a standard to compare different Western blots.
Finally, we would like to thank again the editor and all three reviewers for their thorough and constructive comments, which clearly helped us to improve our manuscript. We hope that it is now suitable for publication in the Nature Communications.